A program of research is being proposed to study the enzymology and molecular biology of DNA replication in human mitochondria. The two origins of mitochondrial DNA replication, OH and OL, function in two distinct mechanisms utilizing different enzymatic machinery. Initiation at OL requires a DNA primase activity whereas initiation at OH appears to be primed by transcripts synthesized by RNA polymerase. Attempts will be made to identify and purify the different components of the DNA primase complex, particularly the primase polypeptide and factors that interact with OL to ensure proper coupling of primase and DNA polymerase activities. Monoclonal antibodies to the DNA primase complex will be prepared and the epitopes will be identified using a variety of functional criteria. It was shown previously that the mitochondrial DNA primase activity of human cells resided in a massive complex that contains small RNA. Identification of the polypeptide components of the DNA primase complex will undoubtedly facilitate understanding of the function of the RNA and the physical structure of the enzyme complex. These studies will ultimately reveal the mechanism of action of the mitochondrial DNA primase. Attempts will also be made to establish an in vitro assay for studying initiation at OH. Mitochondrial lysate will be fractionated to yield an optimal preparation for synthesizing H-strand mitochon- drial DNA on cloned DNA template. The active preparation will be further fractionated and reconstituted in order to identify the enzymatic activities and factors that are involved. This in vitro assay will also be used to analyze structures of the DNA template that are essential features of OH using deletion and mutagenesis techniques. Results of this proposed research will provide insights into the general mechanism of DNA replication in eukaryotic cells. It is hoped that this knowledge will help advance our understanding of the control of cell division and the mechanisms of neoplasia.